Tetracycline compounds for treatment of Cryptosporidium parvum related disorders

ABSTRACT

Methods and compositions for treating  Cryptosporidium parvum  related disorders in a mammal are discussed. Several novel tetracycline compounds useful for treating  Cryptosporidium parvum  related disorders are also included.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/982,728, filedNov. 4, 2004, now U.S. Pat. No. 7,202,235; which is a continuation ofSer. No. 09/768,189, filed Jan. 23, 2001, now U.S. Pat. No. 6,833,365;which claims priority to U.S. Provisional Patent Application Ser. No.:60/178,519, filed on Jan. 24, 2000, the entire contents of each of whichare hereby incorporated herein by reference. The entire contents of eachof the above applications and patents are hereby expressly incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Cryptosporidium parvum (or C. parvum) is an enteric protozoa of thephylum Apicomplexa. It is a major cause of diarrhea in humans andcertain domestic animals (Tzipori, Advances in Parasitology (1988)27:63-129). It is responsible for sporadic cases and major waterborneoutbreaks of self-limiting diarrhea in immunocompetent humans (Current,W. L. et al., Clinical Microbiology Reviews, (1991) 4:325). C. parvum isone of several important opportunistic infections (OI) associated withdiarrhea and wasting in patients with AIDS. Depending on location in theUnited States, some 10 to 15% of individuals with AIDS contract thedisease (Peterson, Clinical Infectious Diseases, (1992) 15:903). Theinfection in the immunodeficient host often becomes persistent, causinglife-threatening, profound, unremitting watery diarrhea and wasting. Aprolonged course of infection often leads to a spread of infection intothe hepatobiliary (HB) tract causing serious complications (Flanigan,Progress in Clinical Parasitiology (1993) 3:1). Of the OI affectingpatients with AIDS, C. parvum is one of only a few infections againstwhich there is no consistently effective treatment. There had been onlya few reports of successful treatment of individual AIDS patients withhyperimmune bovine colostrum (Tzipori, Lancet. (1986) ii:344; Ungar,Gastroenterology (1990) 98:486) and with paromomycin (PRM) (Fitchenbaum,Clinical Infectious Diseases (1993) 16:298). Since none of the availableantimicrobial agents are consistently effective, a search for noveltherapeutic agents against C. parvum is necessary. With increasedsurvival time of patients with AIDS due to much improved patient care,the incidence of the disease in this population is likely to continue torise.

The lifecycle of C. parvum is similar to that of other coccidia whichinfect mammals. The lifecycle can be divided into six majordevelopmental events (Current, Journal of Protozoology, (1986) 33:98);excystation, the release of infective sporozoites; merogony, the asexualmultiplication within host cells; gametogony, the formation of micro andmacrogametes; fertilization, the union of micro and macrogametes; oocystwall formation, to produce an environmentally resistant stage thattransmits infection from one host to another; and sporogony, theformation of infective sporozoites within the oocyst wall. Eachintracellular stage of C. parvum resides within a parasitophorousvacuole confined to the microvillous region of the host cell, whereascomparable stages of Toxoplasma gondii, Eimeria, or Isopora to which C.parvum is closely related, occupy parasitophorous vacuoles deep withinthe host cytoplasm. Oocysts of C. parvum undergo sporogony while theyare within the host cells and are infective when released in the feces.Approximately 20% of the oocysts of C. parvum are thin walled anddischarge their sporozoites within the lumen of the same host, while 80%form a thick two-layered environmentally resistant oocyst wall, and aredischarged in the feces. The four sporozoites emerging from thethin-walled oocysts and repeated cycles of schizogeny contribute to thepersistence of the infection in the immunodeficient host known asautoinfection.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains to a method for controllingCryptosporidium parvum in a mammal, by administering to the mammal aneffective amount of a tetracycline compound. Examples of tetracyclinecompounds of the invention include compounds of formula I:

wherein:

-   -   X is CHC(R¹³Y′Y), CHR⁶, S, NR⁶, or O;    -   R², R⁴ and R^(4′) are each hydrogen, alkyl, alkenyl, alkynyl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,        arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug        moiety;    -   R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug        moiety;    -   R⁵ is hydroxy, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,        heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,        alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;    -   R⁶, R⁷, R⁸ and R⁹ are each independently hydrogen, hydroxyl,        halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   Y′ and Y are each independently hydrogen, halogen, hydroxyl,        cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   and pharmaceutically acceptable salts thereof.

The invention also pertains to a method for treating a Cryptosporidiumparvum related disorder in a mammal, by administering to the mammal aneffective amount of a tetracycline compound. In an embodiment, thetetracycline compound is of formula (I). In another advantageousembodiment, the mammal is immunocompromised, e.g., suffering from AIDSor undergoing chemotherapy. Preferably, the mammal is a human.

In another embodiment, the invention pertains to pharmaceuticalcompositions containing an effective amount of a tetracycline compoundto treat a Cryptosporidium parvum related disorder in a mammal and apharmaceutically acceptable carrier.

In yet another embodiment, the invention features a tetracyclinecompound of the formula:

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains, at least in part to methods for controllingCryptosporidium parvum in a mammal, by administering to the mammal aneffective amount of a tetracycline compound.

Cryptosporidium is a coccidian protozoan parasite that has gained muchattention in the last 20 years as a clinically important human pathogen.For several decades, Cryptosporidium was thought to be a rare,opportunistic animal pathogen, but the first case of humancryptosporidiosis in 1976 involved a 3-year-old girl from ruralTennessee who suffered severe gastroenteritis for two weeks (FlaniganProg Clin Parasitol (1993) 1). Electron microscopic examination of theintestinal mucosa led to the discovery that Cryptosporidium parvum wasthe infectious species in humans. In the early 1980s, the strongassociation between cases of cryptosporidiosis and immunodeficientindividuals (such as those with AIDS—acquired immunodeficiency syndrome)brought Cryptosporidium to the forefront as a ubiquitous human pathogen.Presently, the increasing population of immunocompromised patients andthe various outbreaks of cryptosporidiosis through infection bywater-borne Cryptosporidium oocysts (often in drinking water) havecreated world wide interest in this pathogen. Unlike other intestinalpathogens, Cryptosporidium can infect several different hosts, cansurvive most environments for long periods of time (Keusch, et al.Schweiz Med Wochenschr, (1995) 125(18):899), and inhabit all climatesand locales.

The terms “tetracycline” or “tetracycline derivative” compounds includetetracycline and other tetracycline family members such as,chlortetracycline, oxytetracycline, demeclocycline, methacycline,doxycycline, minocycline, and sancycline. Additional tetracyclinecompounds can be found, for example, in U.S. patent application Ser. No.09/234,847, and U.S. Pat. Nos. 5,834,450; 5,532,227; 5,789,395;5,639,742 and German patents DE 28 14 974 and DE 28 20 983. The entirecontents of the aforementioned applications and patents are herebyexpressly incorporated herein by reference.

More recent research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration; and for developing new tetracyclineanalogues which might prove to be equal or more effective than theoriginally introduced tetracycline families beginning in 1948.Representative of such developments include U.S. Pat. Nos. 3,957,980;3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280; 4,018,889;4,024,272; 4,126,680; 3,454,697; and 3,165,531. These issued patents aremerely representative of the range of diversity of investigationsseeking tetracycline and tetracycline analogue compositions which arepharmacologically active, and the contents of each are expresslyincorporated by reference.

Historically, soon after their initial development and introduction, thetetracyclines, regardless of specific formulation or chemical structure,were found to be highly effective pharmacologically against rickettsiae,a number of gram-positive and gram-negative bacteria, and the agentsresponsible for lymphogranuloma venereum, including conjunctivitis, andpsittacosis. Hence, tetracyclines became known as “broad spectrum”antibiotics. With the subsequent establishment of their in vitroantimicrobial activity, effectiveness in experimental infections, andpharmacological properties, the tetracyclines as a class rapidly becamewidely used for therapeutic purposes. However, this widespread use oftetracyclines for both major and minor illnesses and diseases leddirectly to the emergence of resistance to these antibiotics even amonghighly susceptible bacterial species both conunensal and pathogenic(e.g., pneumococci and Salmonella). The rise of tetracycline-resistantorganisms has resulted in a general decline in use of tetracyclines andtetracycline analogue compositions as antibiotics of choice.

In one embodiment, the tetracycline compound of the invention inhibitsmore than 70% of Cryptosporidium parvum at a concentration less than 100μg/ml, less than 50 μg/ml, less than 20 μg/ml, less than 10 μg/ml, orless than 1 μg/ml. The inhibition of Cryptosporidium parvum can betested using the assay described in Example 2.

Tetracycline compounds of the invention include compounds of Formula I:

wherein:

-   -   X is CHC(R¹³Y′Y), CHR⁶, S, NR⁶, or O;    -   R², R⁴ and R^(4′) are each hydrogen, alkyl, alkenyl, alkynyl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,        arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug        moiety;    -   R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug        moiety;    -   R⁵ is hydroxy, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,        heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,        alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;    -   R⁶, R⁷, R⁸ and R⁹ are each independently hydrogen, hydroxyl,        halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   Y′ and Y are each independently hydrogen, halogen, hydroxyl,        cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or        arylalkyl; and pharmaceutically acceptable salts thereof.

In one further embodiment, R²′, R³, R¹⁰, R¹¹, and R¹² are each hydrogenor a prodrug moiety. Furthermore, R⁴ and R^(4′) can be alkyl, e.g.,lower alkyl, e.g., methyl, ethyl, propyl, or butyl. In anotherembodiment, R⁵ is hydroxyl, hydrogen, or alkanoyl, e.g., an ester,advantageously, a propanoic ester. In yet another embodiment, X is S orCHR⁶. Examples of R⁶ include alkyl groups, e.g., methyl, ethyl, propyl,or halogens or hydroxyl groups. Advantageously, R⁶ may comprise aheteroatom, such as, for example, a sulfur atom. For example, R⁶ may bea thioether, e.g., a cyclopentylthio ether. Advantageous examples of R⁹include hydrogen atoms, and alkyl (e.g., t-butyl) and alkenyl (e.g.,cyclopentenyl) groups.

Tetracycline compounds of the invention include, for example, compoundsof the formulae:

Other examples of preferred tetracycline compounds of the inventioninclude, for example, 5-propionyl-6-cyclopentylsulfanylmethyldoxycycline; thiatetracycline; 9-cyclopent-1-enyl-doxycycline;5-propionyl-9-tert-butyl-doxycycline; doxycycline; 9-tert-butyldoxycycline; 9-cyclohex-1-enylethynyl minocycline; and6-cyclopentylsulfanylmethyl doxycycline.

The tetracycline compounds of the invention can be synthesized using themethods described in Example 1. Scheme 1 depicts a general synthesis ofa thiol ether from methacycline.

13-substituted thiols can be synthesized by the method outlined inScheme 1, above. The synthesis of the compounds is described in greaterdetail in Example 1. Generally,13-substituted thiol ethers (1B) can besynthesized by heating a tetracycline salt (such as methacyclinehydrochloride, 1A), AIBN (2,2′-azobisisobutyronitrile), and a thiol inethanol at reflux for six hours under an inert atmosphere.

9-substituted tetracyclines such as 9-cyclopentenyl doxycycline can besynthesized by the method shown in Scheme 2. As in Scheme 2,9-substituted tetracycline compounds can be synthesized by treating atetracycline compound (e.g., doxycycline, 2A), with sulfuric acid andsodium nitrate. The resulting product is a mixture of the 7-nitro and9-nitro isomers (2B and 2C, respectively). The 7-nitro (2B) and 9-nitro(2C) derivatives are treated by hydrogenation using hydrogen gas and aplatinum catalyst to yield amines 2D and 2E. The isomers are separatedat this time by conventional methods. To synthesize 9-substitutedalkenyl derivatives, the 9-amino tetracycline compound (2E) is treatedwith HONO, to yield the diazonium salt (2F). The salt (2F) is treatedwith an appropriate halogenated reagent (e.g., R⁹Br, wherein R⁹ is anaryl, alkenyl, or alkynyl moiety) to yield the desired compound (e.g.,in Scheme 2, 9-cyclopent-1-enyl doxycycline (2G)).

The term “alkenyl” includes unsaturated aliphatic groups, includingstraight-chain alkenyl groups, branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups, alkenyl substituted cycloalkyl orcycloalkenyl groups, and cycloalkenyl substituted alkyl or alkenylgroups. The term alkenyl further includes alkenyl groups, which canfurther include oxygen, nitrogen, sulfur or phosphorous atoms replacingone or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen,sulfur or phosphorous atoms. In preferred embodiments, a straight chainor branched chain alkenyl group has 10 or fewer carbon atoms in itsbackbone (e.g., C₁-C₁₀ for straight chain, C₃-C₁₀ for branched chain),and more preferably 6 or fewer. Likewise, preferred cycloalkenyl groupshave from 4-7 carbon atoms in their ring structure, and more preferablyhave 5 or 6 carbons in the ring structure, e.g., cyclopentene orcyclohexene.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. The term alkyl further includes alkyl groups,which can further include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone, e.g., oxygen,nitrogen, sulfur or phosphorous atoms. In preferred embodiments, astraight chain or branched chain alkyl has 10 or fewer carbon atoms inits backbone (e.g., C₁-C₁₀ for straight chain, C₃-C₁₀ for branchedchain), and more preferably 6 or fewer. Likewise, preferred cycloalkylshave from 4-7 carbon atoms in their ring structure, and more preferablyhave 5 or 6 carbons in the ring structure.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acyl amino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)).

The term “aryl” includes aryl groups, including 5- and 6-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine,pyrazine, pyridazine and pyrimidine, and the like. Aryl groups alsoinclude polycyclic-fused aromatic groups such as naphthyl, quinolyl,indolyl, and the like. Those aryl groups having heteroatoms in the ringstructure may also be referred to as “aryl heterocycles”, “heteroaryls”or “heteroaromatics”. The aromatic ring can be substituted at one ormore ring positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromaticmoiety. Aryl groups can also be fused or bridged with alicyclic orheterocyclic rings which are not aromatic so as to form a polycycle(e.g., tetralin).

The terms “alkenyl” and “alkynyl” include unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. Examples of substituents of alkynyl groups include, forexample alkyl, alkenyl (e.g., cycloalkenyl, e.g., cyclohenxenyl), andaryl groups.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto three carbon atoms in its backbone structure. Likewise, “loweralkenyl” and “lower alkynyl” have similar chain lengths.

The terms “alkoxyalkyl”, “polyaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (includingalkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “alkylsulfinyl” include groups which have one or more sulfinyl(SO) linkages, typically 1 to about 5 or 6 sulfinyl linkages.Advantageous alkylsulfinyl groups include groups having 1 to about 12carbon atoms, preferably from 1 to about 6 carbon atoms.

The term “alkylsulfonyl” includes groups which have one or more sulfonyl(SO₂) linkages, typically 1 to about 5 or 6 sulfonyl linkages.Advantageous alkylsulfonyl groups include groups having 1 to about 12carbon atoms, preferably from 1 to about 6 carbon atoms.

The term “alkanoyl” includes groups having 1 to about 4 or 5 carbonylgroups. The term “aroyl” includes aryl groups, such as phenyl and othercarbocyclic aryls, which have carbonyl substituents. The term “alkaroyl”includes aryl groups with alkylcarbonyl substituents, e.g.,phenylacetyl.

The structures of some of the tetracycline compounds of this inventioninclude asymmetric carbon atoms. The isomers arising from the chiralatoms (e.g., all enantiomers and diastereomers) are included within thescope of this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis.

The invention also pertains to a pharmaceutical composition containingan effective amount of a tetracycline compound to treat or prevent aCryptosporidium parvum related disorder in a mammal and apharmaceutically acceptable carrier. The pharmaceutical composition maycontain an effective amount of a supplementary anti-Cryptosporidiumparvum agent.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allows the tetracycline compounds to perform their intendedfunction, e.g., treating a Cryptosporidium parvum related disorder orpreventing a Cryptosporidium parvum related disorder. Examples of suchcarriers include solutions, solvents, dispersion media, delay agents,emulsions and the like. The use of such media for pharmaceuticallyactive substances are well known in the art. Any other conventionalcarrier suitable for use with the tetracycline compounds of the presentinvention are included.

For example, one or more compounds of the invention may be administeredalone to a subject, or more typically a compound of the invention willbe administered as part of a pharmaceutical composition in mixture withconventional excipient, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, oral or otherdesired administration and which do not deleteriously react with theactive compounds and are not deleterious to the recipient thereof.Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohol, vegetable oils, polyethyleneglycols, gelatin, lactose, amylose, magnesium stearate, talc, silicicacid, viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

At least many of the tetracycline compounds of the invention suitablymay be administered to a subject in a protonated and water-soluble form,e.g., as a pharmaceutically acceptable salt of an organic or inorganicacid, e.g., hydrochloride, sulfate, hemi-sulfate, phosphate, nitrate,acetate, oxalate, citrate, maleate, mesylate, etc. Also, where anappropriate acidic group is present on a compound of the invention, apharmaceutically acceptable salt of an organic or inorganic base can beemployed such as an ammonium salt, or salt of an organic amine, or asalt of an alkali metal or alkaline earth metal such as a potassium,calcium or sodium salt.

Therapeutic compounds can be administered to a subject in accordancewith the invention by any of a variety of routes. Topical (includingtransdermal, buccal or sublingual), and parenteral (includingintraperitoneal, subcutaneous, intravenous, intradermal or intramuscularinjection) are generally preferred.

For parenteral application, particularly suitable are solutions,preferably oily or aqueous solutions as well as suspensions, emulsions,or implants, including suppositories. Therapeutic compounds will beformulated in sterile form in multiple or single dose formats such asbeing dispersed in a fluid carrier such as sterile physiological salineor 5% saline dextrose solutions commonly used with injectables.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

For topical applications, the tetracycline compound(s) can be suitablyadmixed in a pharmacologically inert topical carrier such as a gel, anointment, a lotion or a cream. Such topical carriers include water,glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides,fatty acid esters, or mineral oils. Other possible topical carriers areliquid petrolatum, isopropylpalmitate, polyethylene glycol, ethanol 95%,polyoxyethylene monolauriate 5% in water, sodium lauryl sulfate 5% inwater, and the like. In addition, materials such as anti-oxidants,humectants, viscosity stabilizers and the like also may be added ifdesired.

The actual preferred amounts of active compounds used in a given therapywill vary according to the specific compound being utilized, theparticular compositions formulated, the mode of application, theparticular site of administration, etc. Optimal administration rates fora given protocol of administration can be readily ascertained by thoseskilled in the art using conventional dosage determination testsconducted with regard to the foregoing guidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

The language “effective amount” of the tetracycline compound is thatamount necessary or sufficient to control Cryptosporidium parvum in amammal, e.g., prevent the various morphological and somatic symptoms ofa Cryptosporidium parvum-related disorder. The effective amount can varydepending on such factors as the size and weight of the subject, thetype of illness, or the particular tetracycline compound. For example,the choice of the tetracycline compound can affect what constitutes an“effective amount”. One of ordinary skill in the art would be able tostudy the aforementioned factors and make the determination regardingthe effective amount of the tetracycline compound without undueexperimentation. An in vivo assay as described in Example 4 below or anassay similar thereto (e.g., differing in choice of cell line or type ofillness) also can be used to determine an “effective amount” of atetracycline compound. The ordinarily skilled artisan would select anappropriate amount of a tetracycline compound for use in theaforementioned in vivo assay. Preferably, the effective amount of thetetracycline is effective to treat a mammal suffering from aCryptosporidium parvum related disorder.

The term “mammal” includes animals which are capable of having aCryptosporidium parvum related disorder. Examples of mammals include,but are not limited to, ruminants (e.g., cattle and goats), mice, rats,hamsters, dogs, cats, horses, pigs, sheep, lions, tigers, bears,monkeys, chimpanzees, and, in a preferred embodiment, humans. The mammalmay be immunocompetent or immunocompromised, e.g., suffering from animmunodeficiency. For example, the mammal may have AIDS or may havepreviously or concurrently undergone chemotherapy. In anotherembodiment, the mammal may be elderly or young. The mammal may or maynot be suffering from a Cryptosporidium parvum related disorder. Thetetracycline compounds may be administered to a mammal susceptible to aCryptosporidium parvum disorder to prevent the occurrence of thedisorder.

The language “Cryptosporidium parvum related disorder” includesdisorders which are related to the infection or the presence ofCryptosporidium parvum in a mammal. Examples of Cryptosporidium parvumrelated disorders include diarrhea and cryptosporidiosis.

In another embodiment, the invention pertains to a method for treating aCryptosporidium parvum related disorder in a mammal, by administering tothe mammal an effective amount of a tetracycline compound such that saidmammal is treated for the disorder.

In a further embodiment, the invention includes the administration of asupplementary anti-Cryptosporidium parvum agent in combination with thetetracycline compound of the invention.

The language “in combination with” includes simultaneous administrationof the tetracycline compound of the invention and the supplementaryanti-Cryptosporidium parvum agent, administration of the agent first,followed by the tetracycline compound and administration of thetetracycline compound first, followed by the agent. The invention alsoincludes the administration of other therapeutic agents in combinationwith the tetracycline compounds of the invention. For example, thetetracycline compounds of the invention may be administered incombination with drugs used in AIDS therapy for AIDS patients.

The term “supplementary agent” includes compounds known in the art tohave anti-Cryptosporidium parvum activity such as, for example,paromomycin and derivatives thereof.

The present invention is further illustrated by the following examples.These examples are provided to aid in the understanding of the inventionand are not to be construed as limitations thereof.

EXEMPLIFICATION OF THE INVENTION Example 1 Synthesis of TetracyclineCompounds

The following example discusses methods of synthesizing the tetracyclinecompounds of the invention.

Experimental

Melting points were taken on a Mel-Temp capillary melting pointapparatus and are uncorrected. Nuclear magnetic resonance (¹H NMR)spectra were recorded at 300 MHz on a Bruker Avarice spectrometer. Thechemical shift values are expressed in δ values (ppm) relative totetramethylsilane or 3-(trimethylsilyl)-1-propanesulfonic acid, sodiumsalt, as either an internal or external standard using CDCl₃, DMSO-d₆,or MeOH-d₄ as the solvent. Column chromatography was performed accordingto the method of Still using Baker “flash” grade silica gel (40 μm) thatwas treated with a saturated solution of Na₂EDTA, washed with water,filtered and dried in an oven at 130° C. for three hours prior to use.Analytical TLC separations employed the use of 0.25 mm silica gel plateswith florescence indicator obtained from J.T. Baker Chemical Co.,Phillipsburg, N.J., that were pretreated by immersion into a saturatedsolution of Na₂EDTA for five minutes and reactivated at 130° C. forthree hours. Solvent systems used were as follows: 50:50:5 CHCl₃/MeOH/5%Na₂EDTA (lower phase) (I), 65:20:5, CHCl₃/MeOH/Na₂EDTA (lower phase)(II). Visualization of TLC was accomplished by 0.5% aqueous Fast Blue BBsalt and heating at 130° C. for 5 minutes. Analytical HPLC was performedon a Waters Bondapak C18 reverse phase column by using two Varian SD 100HPLC pumps at a 1.6 mL/min flow rate controlled by software. Detectionwas by UV absorption with Model 441 absorbance detector operating at 280nm. Mobile phases used followed a linear gradient from 30% to 100%methanol over 30 minutes at 1.6 mL/min flow rate followed by isocraticelution with MeOH; solvent system A: 0.02 M Na₂HPO₄+0.001 M Na₂EDTAadjusted to pH 4.5 with H₃PO₃; solvent system B: 100% MeOH.Semipreparative HPLC separations used a Waters semipreparative C18reverse-phase column at a flow rate of 6.4 mL/min. Low and highresolution mass spectra were performed on a PE Mariner spectrometer(Nelson et al., J. Med. Chem. (1993) 36(3):374).

General Procedure for the Synthesis of 13-[(Substitutedphenyl)thio]-5-hydroxy-6-deoxytetracyclines13-(Phenylthio)-5-hydroxy-6-α-deoxytetracycline

A mixture of methacycline hydrochloride (3.0 g, 6.2 mmol), AIBN (250mg), and thiophenol (1.32 g, 12.4 mmol) in ethanol (50 mL) was heated atreflux for 6 hours while under N₂. The reaction mixture was cooled,filtered to remove insolubles, and concentrated to one-fifth volumeunder reduced pressure. Precipitation of the resulting solution in coldEt₂O led to isolation of crude-product (2.17 g). The solid was dissolvedin hot H₂O, and extracted into CHCl₃ at pH 5.0. Removal of the solid andtreatment with activated charcoal in MeOH led to isolation of theproduct (0.958 g, 27.1%): mp=164-171° C.; TLC R_(f)=0.67 (I); HPLCt_(R)=14.45 min; ¹H NMR (CDCl₃) δ 11.9 (br s), 9.3 (br s), 7.35 (m, 6H), 6.83 (d, 1H), 6.74 (d, 1H), 5.95 (br s, 1H), 4.10 (br s, 1H), 3.82(s, 2H), 3.60 (br s, 1H), 3.10 (m, 2H), 2.60 (m, 1H), 2.48 (s, 6H); MS(FAB) m/z 552, 553 ([M+H]+); 445(M-C₆H₅—S+H).

13-(Cyclopentylthio)-5-hydroxy-6-α-deoxytetracycline

Methacycline hydrochloride (5.0 g, 10.4 mmol) was placed in around-bottomed flask and suspended in 100 mL of ethanol. Twentymilliliters of cyclopentanethiol (0.0270 mol) and AIBN (250 mg) wereadded, and the reaction mixture was refluxed with stirring for 12 hourswhile under N₂. The mixture was reduced to one-fifth volume bydistillation and filtered. The filtrate was dripped slowly into coldEt₂O with stirring, resulting in the formation of a yellow precipitate.The compound was purified further by either column chromatography onEDTA-silica, by extraction at pH 4.5 into CH₂Cl₂, or by HPLCchromatography. An analytical sample was produced by HPLC as a yellowsolid in moderate yield (28.3%). Higher yields were obtained by theextraction method and treatment with activated charcoal in MeOH (32.1%).mp=132-139° C.; TLC R_(f)=0.80 (I); HPLC t_(R)=21.19 min; ¹H NMR(MeOH-d₄) δ 7.38 (t, 1H), 7.02 (d, 1H), 6.72 (d, 1H), 4.10 (s, 2H), 2.70(br s, 6H), 1.81-2.01 (br m, 2 H), 1.28-1.75 (br m, 6H); HRMS (FAB)calculated for C₂₇H₃₂N₂O₈S 545.1957 (M+1), found 545.1960 (M+1).

[4S-(4α,12aα)]-9-(nitro)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide

To an ice cold solution of 1.0 g of doxycycline hydrochloride in 10 mlof concentrated sulfuric acid was added 0.231 g of potassium nitrate.The reaction was stirred for 1 hour under ambient atmosphere. Themixture was then poured over 150 g of ice and the resulting solid wasextracted with n-butanol and dried to afford 0.9 g of the desiredproduct as a yellow-green solid. MS(FAB): m/z 490 (M+H). ¹H NMR (CD₃OD):δ 7.50 (d, 1H, J=8.07 Hz, H-8); 6.86 (d, 1H, J=8.07 Hz, H-7); 4.44 (bs,1H, H-4); 3.62 (dd, 1H, J=11.42; 8.35 Hz, H-5); 2.95 (bs, 6H, NMe₂);2.81 (d, 1H, J=11.45 Hz, H-4a); 2.71 (dq, 1H, J=12.41; 6.5 Hz, H-6);2.53 (dd, 1H, J=12.23; 8.20 Hz, H-5a); 1.51 (d, 3H, J=6.78 Hz, CH₃).

[4S-(4α,12aα)]-9-(amino)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide

Into a 200 ml hydrogenation bottle is added 1.0 g of compound 1, 40 mlof methanol, 1 ml of concentrated HCl, and 100 mg of 10% palladium oncarbon. Using a hydrogenation apparatus, the mixture is subjected to 30psi of hydrogen for 3 hours. The catalyst is filtered off and thefiltrate is dried to afford 0.9 g of the dihydrochloride as a yellowsolid. MS(FAB): m/z 460 (M+H). ¹H NMR (CD₃OD): d 7.54 (d, 1H, J=8.08 Hz,H-8); 6.88 (d, 1H, J=8.08 Hz, H-7); 5.16 (dd, J=10.44; 7.94 Hz, H-5);4.44 (bs, 1H, H-4); 3.74 (d, 1H, J=2.07 Hz, H-4); 3.04 (bs, 6H, NMe₂);2.90 (dd, 1H, J=7.94; 2.07 Hz, H-4a); 2.72 (dq, 1H, J=12.31; 6.56 Hz,H-6); 2.61 (dd, 1H, J=12.31; 10.44 Hz, H-5a); 2.54 (q, 2H, J=7.48 Hz,CH₂—C); 1.44 (bs, 9H, CMe₃); 1.29 (d, 3H, J=6.56 Hz, CH₃); 1.20 (t, 3H,J=7.48 Hz, C—CH₃).

[4S-(4α,12aα)]-9-(diazonium)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide

A 10 ml roundbottom flask was charged with 100 mg of compound 2 anddissolved in 4 ml of 0.1 N methanolic hydrochloric acid. The solutionwas cooled to 0° C. and 35 μl of butyl nitrite was added with stirring.After 1 hour, the bright red reaction mixture was added dropwise to 100ml of cold anhydrous diethyl ether. The product was collected byfiltration, washed with ether, and dried in a vacuum dessicator to give73 mg of the diazonium salt as an orange solid. MS(FAB): m/z 472 (M+H).¹H NMR (CD₃OD): d 7.55 (d, 1H, J=8.08 Hz, H-8); 6.86 (d, 1H, J=8.08 Hz,H-7); 5.13 (dd, J=10.44; 7.94 Hz, H-5); 4.41 (bs, 1H, H-4); 3.72 (d, 1H,J=2.07 Hz, H-4); 3.04 (bs, 6H, NCH₃); 2.90 (dd, 1H, J=7.94; 2.07 Hz,H-4a); 2.70 (dq, 1H, J=12.31; 6.56 Hz, H-6); 2.61 (dd, 1H, J=12.31;10.44 Hz, H-5a); 2.2 (m, 6H, J=7.48 Hz, Acetyl); 1.44 (bs, 9H, C(CH₃)₃);1.29 (d, 3H, J=6.56 Hz, CH₃); 1.20 (t, 3H, J=7.48 Hz, C—CH₃).

General Procedure for Olefination

To a solution of 0.1 g of a 9-diazonium compound in wet methanol isadded 0.05 equivalents of palladium acetate. The reaction mixture isstirred for 5 minutes at room temperature, and 2 equivalents of thedesired olefin is added. Stirring is continued for 18 hours underambient atmosphere or followed by HPLC. Upon completion, the catalyst isfiltered off and the filtrate dried to give the crude product. Thepurified product is isolated by preparative reverse-phase HPLC usingmethanol and phosphate buffer gradient.

9-(1′-cyclopentenyl)minocycline

MS(FAB): m/z 511 (M+H).

Example 2 Assay for the Ability of Tetracycline Compounds to ControlCryptosporidium parvum Infection in Vitro

This assay is designed to test the ability of a tetracycline compound tocontrol Cryptosporidium parvum infection in vitro. The results show thattetracycline compounds of the invention can be used to control thegrowth of C. parvum.

MDCK cells were grown in 96-well microtiter plastic plates, and wereseeded with approximately 5×10⁴ cells per well, using DMEM as thegrowth/maintenance medium with 10% FCS. Plates were normally grown toconfluence 2-3 days after having been seeded with bleached, less thanfour week-old, C. parvum oocysts. The C. parvum isolate used most oftenwas the human-derived, calf-propagated GCHI (Tzipori, Clin. Diagn. Lab.Immunol. (1994) 1:450; Tzipori, J. Infect. Dis. (1995) 172:1160). TheDMEM medium was also added to the negative control wells. Thetetracycline compounds and the oocysts were added to the wellsconcurrently. The cells were subsequently monitored twice daily and anyapparent morphologic changes were recorded. The monolayers were fixedwith methanol after 48 hour incubation at 37° C. in 8% CO₂.

Table 1 shows the Inhibition assay results of the tetracycline compoundsin MDBK infected cells. The tetracycline compounds were used at variousconcentrations (0.1-2000 μg/mL) and were dissolved directly into theculture medium. A conventional indirect immunofluorecensce (IF) assaywas used to detect and enumerate parasite forms after 48-hourincubation. For the purposes of this assay, a polyclonal rabbitanti-sporozoite antibody was produced. It was used at a dilution of1:1000 and does not react with oocyst shells. The secondary antibody, afluoroscein-conjugated goat anti-rabbit IgG, was used at a concentrationof 1:100, according the manufacturer's instructions. The primaryantibody was added after fixation for thirty minutes and after vigorouswashing, the secondary antibody was incubated for another thirtyminutes. The dried microtiter plate was viewed with an invertedmicroscope (×10 mag.), under ultraviolet light. For parasite counts, asemi-automated video imaging MCID system was used to facilitate theenumeration and analysis of the parasite data. In Table 1, goodinhibition of C. parvum is indicated by ‘*’ (e.g., 70% inhibition atconcentrations above 10 μg/ml and above) and very good inhibition of C.parvum is indicated by ‘**’ (e.g., 70% inhibition at concentrationsbelow 10 μg/ml). Some compounds of the invention have 70% inhibition atconcentrations below 1 μg/ml.

The study shows that all of the tetracycline compounds are capable ofinhibiting or decreasing the amount C. parvum in a sample of cells.

TABLE 1 Compound Inhibition

**

**

*

*

**

*

**

**

Example 3 In Vitro Cytotoxicity Assay of Tetracycline Compounds

The following assay is designed to test the cytotoxicity of thetetracycline compounds of the invention on MDBK cells. Advantageouscompounds of the invention are compounds with low cytotoxicity.

Cytotoxicity of the tetracycline compound is measured by the Cell Titer96™ Aqueous, a non-radioactive cell proliferation assay, available as acommercial kit. It is a colorimetric method for determining the numberof viable cells in proliferation or chemosensitivity assays. The assayis performed by growing MDBK cells in 96-well microliter plates, as inExample 2. Once confluent, the media is aspirated and replaced with 200μL of media containing the tetracycline compound concentrations whichwere tested in Example 2. After 48 hour incubation, 40 μL/well offreshly prepared MTS/PMS solution is added. The plate is incubated fortwo hours at 37° C. and 8% CO₂ and then 100 μL of supernatant from eachwell is transferred to a new 96-well plate. The optical density isdetermined at 490 nm by an ELISA plate reader and the results arerecorded and analyzed. Percent toxicity is calculated by subtracting themean optical density (OD) of the medium control supernatants (notetracycline compound) by the mean OD of the tetracycline compoundsupernatants and dividing by the OD of the medium control andmultiplying by 100.

Example 4 In Vivo Assay of Inhibition of Cryptosporidium parvumInfection

This study is designed to test the ability of a tetracycline compound tocontrol Cryptosporidium parvum infections in mice. Advantageouscompounds of the invention control the Cryptosporidium parvum infectionwithout killing the mice.

Three 4-week old C.B-17 SCID mice are randomized into six groups ofseven mice each. Each animal receives a single I.P. injection of 1 mg ofXMG 1.2 mAb. Two hours later, mice in five of the six groups areinfected with 10⁷ GCH1 oocysts via oral inoculation. Treatment with atetracycline compounds begins on day 6, post infection, in two divideddoses/day and continues for 10 days.

At the end of the experiment, all animals are necropsied and sectionsare taken from the pyloric region of the stomach, mid small intestine,terminal ileum, cecum, proximal colon, and liver/gall bladder forhistological analysis to determine the extent of mucosal infection. Eachsite is assigned a score depending upon the extent of the infection. Inthis system, scores range from 0 (no infection) to 5 (extensiveinfection). Data is presented as the mean total score of the five sites.Oocyst shedding is monitored in all infected animals three times perweek, beginning on day 4 of infection. Body weights are determined onceper week throughout the course of study.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, issued patents, andpublished patent applications cited throughout this application arehereby incorporated by reference. The appropriate components, processes,and methods of those patents, applications and other documents may beselected for the present invention and embodiments thereof.

1. A method for controlling Cryptosporidium parvum in a mammal whereinthe presence of Cryptosporidium parvum results in a Cryptosporidiumparvum related disorder in said mammal, comprising administering to saidmammal an effective amount of a tetracycline compound, such thatCryptosporidium parvum is controlled in said mammal, wherein saidtetracycline compound comprises the following formula (I)

wherein X is CHC(R¹³Y′Y), CHR⁶, S, NR⁶, or O; R², R⁴ and R^(4 ′) areeach hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, heterocyclic orheteroaromatic; R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen; R⁵ ishydroxyl, hydrogen, thiol, alkaroyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R⁸ and R⁹ areeach independently hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, or an arylalkyl; R⁶ is hydrogen, hydroxyl, halogen, thiol,substituted alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R⁷ ishydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl or an arylalkyl; R¹³ ishydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; Y′ and Y areeach independently hydrogen, halogen, hydroxyl, cyano, sulfhydryl,amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl; or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1, wherein thetetracycline compound is selected from the group consisting of:


3. The method of claim 1, wherein said mammal is immunocompetent.
 4. Themethod of claim 1, wherein said mammal is immunocompromised.
 5. Themethod of claim 1, wherein said mammal is a human.
 6. The method ofclaim 5, wherein said human has an immunodeficiency.
 7. The method ofclaim 5, wherein said human has AIDS.
 8. The method of claim 5, whereinsaid human has undergone chemotherapy.
 9. The method of claim 1, whereinsaid Cryptosporidium parvum related disorder is diarrhea.
 10. The methodof claim 1, wherein said Cryptosporidium parvum related disorder iscryptosporidiosis.
 11. A method for treating a Cryptosporidium parvumrelated disorder in a mammal, comprising administering to said mammal aneffective amount of a tetracycline compound, wherein said tetracyclinecompound comprises the following formula (I)

wherein X is CHC(R¹³Y′Y), CHR⁶, S, NR⁶, or O; R², R⁴ and R^(4′) are eachhydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, heterocyclic or heteroaromatic;R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen; R⁵ is hydroxyl,hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, or an arylalkyl; R⁸ and R⁹ are each independently hydrogen,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R⁶is hydrogen, hydroxyl, halogen, thiol, substituted alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, or an arylalkyl; R⁷ is hydrogen, hydroxyl, halogen, thiol,alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl or an arylalkyl; R¹³ is hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, or an arylalkyl; Y′ and Y are each independently hydrogen,halogen, hydroxyl, cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; or a pharmaceutically acceptable salt thereof, such that saidmammal is treated for said disorder.
 12. The method of claim 11, whereinsaid mammal is immunocompetent.
 13. The method of claim 11, wherein saidmammal is immunocompromised.
 14. The method of claim 11, wherein saidmammal is a human.
 15. The method of claim 14, wherein said human has animmunodeficiency.
 16. The method of claim 14, wherein said human hasAIDS.
 17. The method of claim 14, wherein said human has undergonechemotherapy.
 18. The method of claim 11, wherein said Cryptosporidiumparvum related disorder is diarrhea.
 19. The method of claim 11, whereinsaid Cryptosporidium parvum related disorder is cryptosporidiosis. 20.The method of claim 11, further comprising the administration of apharmaceutically acceptable carrier.
 21. The method of claim 11, furthercomprising the administration of a supplementary anti-Cryptosporidiumparvum agent.
 22. The method of claim 21, wherein said supplementaryagent is paromomycin.
 23. The method of claim 11, wherein saidtetracycline compound is selected from the group consisting of5-propionyl-6-cyclopentylsulfanylmethyl doxycycline; thiatetracycline;6-cyclopentylsulfanylmethyl doxycycline and pharmaceutically acceptablesalts thereof.